After hemorrhagic stroke, subsequent lysis of red blood cells releases toxic levels of heme and then a dangerous excess of free iron in the brain. Hemopexin (HPX) not only binds to excess heme in the blood, but may signal a genetic response to adaptively modulate intracellular iron homeostatic pathways in the brain to adapt to hemorrhagic stresses. We are assessing how HPX controls Iron-regulatory Protein (IRP1 and IRP2) dependent post-transcriptional expression of ferritin (Ftn, for safe iron storage), and transferrin receptor (TfR for iron uptake) in order to optimize iron homeostasis pathways to better protect brain neurons. Critically, we will determine how HPX modifies the IRP regulatory network to adaptively control neuronal translation of ferroportin (for iron export) and that of its binding partner, the amyloid precursor protein (APP). Our lab showed that APP(s) provides significant and novel protective ferroxidase action that also facilitates ferroportin dependent efflux of excess iron from vulnerable neurons. Upon iron influx, IRP1 and IRP2 dissociate from IREs thus releasing Ftn and APP from a translational block for safe storage of iron. Thus our goal is to determine how hemopexin favorably resets this IRE-mediated IRP1/IRP2 control of APP and ferritin gene expression and thereby promotes safe neuronal iron export and storage. Both ferritin and APP confer cytoprotection as active ferroxidases converting Fe2+ to its storage form of Fe3+. To start this reaction excess iron binds REXXE domain in secreted APP(s) and then oxidizes and safely transports/stores it in ferritin. Here, we will test the sufficiency of ths pathway to protect neurons against heme-aggravated damage. We will set up the experimental model of collagenase injection to experimentally induce hemorrhage. It will then be possible to address how hemopexin induces these neuroprotective responses against excess iron from heme by modulating the activity of the REXXE domain in APP and via the iron- responsive elements (IRE) stem loops in the 5'untranslated regions of APP and ferritin mRNAs. ' PUBLIC HEALTH RELEVANCE: This research will provide fundamental information about how iron homeostasis in human brain cells is affected by the hemopexin, which is the known protective scavenger of toxic heme as released after hemorrhagic stroke. We will seek direct evidence that hemopexin can generate signals via iron regulatory proteins to APP and ferritin to better protect to neurons from iron excess. We believe that the novel ferroxidase activity imparted by secreted APP and cellular ferritin mediates this protection to neurons at an accelerated rate when hemopexin is present.